PROJECT SUMMARY/ABSTRACT The goal of the proposed research is to determine the efficacy of Bromodomains and ExtraTerminal (BET) inhibition targets cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC) using in vitro and in vivo models. CSCs are resistant to conventional therapies, leading to tumor propagation and relapse. They are also believed to remain after cancer therapy to initiate the metastatic process. Therefore, specifically targeting CSCs may become a more efficient strategy in HNSCC therapy. Our preliminary data suggests that the BET inhibitors, JQ1 and I-BET762, strongly suppress the self-renewal and chemoresistance properties of CSCs in HNSCC. Of note, these inhibitors profoundly suppressed the expression of a subset of NF-?B and STAT3 target genes, such as IL6. Mechanistically, we found that BRD4, one of the BET family members, interacts with NF-?B p65 on the IL6 promoter and BET inhibition the recruitments of both p65 and BRD4 to the IL6 promoter. Taken together, these results suggest that BET inhibition may target the NF-?B/IL6/STAT3 axis, which is a requirement for the maintenance of CSCs. New studies suggest that BRD4 associates with super- enhancers at key oncogenes in cancers. Cancer cells are reliant on the high expression of these key oncogenes driven by super-enhancers to maintain their malignant phenotype. BET inhibitors could strongly suppress the expression of these key genes and consequent malignant phenotype through releasing BET from super- enhancers. A recent report indicates that NF-?B associates with BET to form super-enhancers at specific genes to the activate inflammation in endothelial cells. JQ1 strongly suppresses this activation through releasing BET from super-enhancers. This highlights that NF-?B may maintain CSC characteristics through association with BET proteins to form super-enhancers at key oncogenic genes. Suppression of BET may thereby target CSCs through disrupting these super-enhancers. Therefore, we hypothesize that BET inhibition releases NF-?B, STAT3 and BRD4 from super-enhancers at genes that determine the characteristics of CSCs to attenuate their properties in HNSCC. This hypothesis will be verified by the following specific aims: (1) To determine BET inhibition suppresses the characteristics of CSCs in HNSCC both in vitro and in vivo. (2) To elucidate the molecular mechanism by which BET inhibition targets CSCs in HNSCC through disrupting super-enhancers. Summary: The completion of the proposed studies will not only provide practical suggestion of BET inhibition in HNSCC therapy but also serve as the basis for subsequent R01 grant proposal. In future, we have two major directions. Based on this study we will form the conceptual framework for a novel therapeutic approach that targeting CSCs in combination with conventional chemotherapy to improve the clinical outcome of HNSCC. We aim to investigate additional genomic targets and pathways by which BET inhibition targets CSCs uncovered by genome wide analysis methods described in this proposal.